Proteins and peptides have the potential to be valuable prophylactic and therapeutic agents in humans and other animal subjects. However, because proteins and peptides are larger and more complex than conventional organic and inorganic drug molecules, the formulation and delivery of such agents present unique problems. In this regard, potentially beneficial protein and peptide drugs typically require the maintenance of their conformational integrity in order to be efficacious with regard to their desired biological properties against the intended targets. A protein's conformation can be altered by any of the numerous protein degradation pathways present in the body.
While there have been extensive and ongoing research efforts focused on novel ways to successfully deliver protein and peptide drugs to their intended targets, effective delivery techniques for these agents have not been perfected.
The present invention relates to the design and development of new types of biological carriers for efficient delivery of medically useful peptides and proteins. These carriers are designed for time-controllable, rapid and tissue-targeted release of the reagents. The strategies to acquire these properties are to construct the carriers from heme biosynthesis deficient trypanosome mutants (Sah et al, 2002). They are transgenically modified, making them responsive to external signals to induce accumulation of porphyrins. The porphyric state mediates cytolysis of the mutant “carriers,” thereby releasing pharmaceutically useful proteins over-expressed by them via pre-transfection with relevant genes of interest. The inherent tissue-specific infection of trypanosomes is exploited for targeting.
Porphyrins are metabolic intermediates in the biosynthesis pathway of heme (Sassa and Nagai 1996). Heme is an indispensable component required for respiration to produce energy in all aerobic organisms, including humans. All porphyrin intermediates formed in this pathway, i.e. uroporphrynogen I, uroporphyrinogen III, coproporphyrinogen III, protoporphyrinogen IX, and protoporphyrin IX are also inherently cytotoxic, especially on exposure to UV irradiation. This results in the generation of free radicals due to photosensitivity of porphyrins. Abnormal accumulation of porphyrins has been reported to result from a dysfunctional heme biosynthesis pathway, leading to human diseases known as “porphyria.” (Sassa 2000). Porphyric individuals suffer from photophobia, tissue necrosis, organ failure and other related systemic disorders. There are different forms of human porphyria, caused by the accumulation of different porphyrin species, resulting from genetic defects of different porphyrin metabolizing enzyme as well as their inhibition by environmental poisons, such as lead.
Application of porphyrins or their precursor, delta-aminolevulinate (ALA) alone is considered to have therapeutic potentials against microbial infection and tumors, especially when this is followed by UV irradiation (Wainwright 1998; Friesen et al. 2002). These procedures have been shown to kill certain pathogenic microorganisms and, more recently, tumor cells in “photodynamic therapy.” In the latter cases, porphyrins are either administered exogenously or induced endogenously within the tumor cells using ALA, the product of ALA-synthase (ALAS)—the first of the eight enzymes in the heme biosynthesis pathway of mammals. The porphyric state generated by either way is non-targeted and transient, and the level of porphyrin accumulation is relatively low. This is due to the substrate “flow-through” and/or feedback and allosteric inhibition in the presence of a complete heme biosynthesis pathway in all aerobic organisms.
Numerous biological carriers of microbial origin have been constructed to deliver drugs and vaccines for potential medical applications (Mollenkopf et al. 2001; Edgeworth et al 2002). Induction of porphyria in microbial carriers for self-destruction presents itself as a potential strategy to achieve simultaneous elimination of the carrier and effective drug release. The feasibility of this strategy is significantly enhanced by using appropriate microorganisms as the carriers with the following inherent and/or genetically modifiable properties: (1) high producer of porphyrins in response to external signals to develop porphyria for time-controllable and rapid cylolysis; (2) mammalian cell, tissue or organ affinity for site-specific “homing” delivery; and (3) ease of in vitro cultivation and transfection of these microbial carriers with available plasmids or other vectors to express foreign genes. The microorganisms with these properties include trypanosomatid protozoa, such as Leishmania spp.
The present invention exploits the virtual absence of heme biosynthesis pathway in trypanosomes to identify or construct their genetic mutants for time-controllable induction of an intense and sustained porphyric state, making it possible to consider their use for targeted release of pharmaceutically important peptides and/or proteins. This is the unique aspect of the present invention.
There has been no similar concept and methodology developed previously with this group of organisms for such applications. The closest materials generated previously are suicidal mutants of Leishmania, but they were constructed not with genes in the heme pathway, but by negative selections for virulence gene knockouts (Titus et al. 1995; Alexander et al. 1998; Gourley et al. 2001; Papadopoulou et al. 2002) or by reverse genetics using widely publicized schemes, e.g., transfection with thymidine kinase gene for responsiveness to ganciclovir as the trigger (LeBowitz et al. 1992). These suicidal mutants also incorporate no time-controllable elements, as designed in the present invention.
Other materials peripherally related to the constructs of the present invention are knockout mutants of individual genes encoding porphyrin metabolizing enzymes in single cell organisms, e.g., algae, yeasts (Kurlandzka et al. 1991; Glerum et al. 1996) and Chlamydomonas (Wang et al. 1975). These mutants have been used for isolation of specific porphyrin species, but have not been considered for use as drug delivery vehicles. Creation of an additional mutation in these engineered or natural mutants for an ALAS negative phenotype is theoretically possible to render them conditional to exogenous ALA for developing porphyria. Such mutant living organisms, are not mammalian cell-, tissue- or organ-specific. Remotely relevant are single experimental steps used in photodynamic therapy of tumors, i.e., ALA induction of these cells to develop a transient porphyric state (Abels et al. 1997; Gibson et. At 1999) and direct administration of porphyrins (Afonso et al. 1990) or other chromogens followed by UV irradiation (Spikes and Bommer 1993) or laser phototherapy (Castro et al. 1996). The aim of photodynamic therapy in these schemes is to use porphyrins for treatment, but not drug delivery as is intended in the present invention.